Kinetic mechanism of the hydrogen-oxidizing hydrogenase from soybean nodule bacteroids

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Abstract
The kinetic mechanism of the unidirectional H2-oxidizing hydrogenase from soybean nodule bacteroids [Rhizobium japonicum] was investigated with highly purified enzyme. Measurements of the Km for H2 vary from 0.97-2.6 .mu.M and the Km for methylene blue varies from 6-17 .mu.M. With H2 and methylene blue as substrates, the initial velocity patterns are intersecting. High levels of methylene blue are inhibitory (KI = 2.4 mM); the inhibition is competitive vs. H2. CO is a competitive inhibitor of H2 (KI = 157 .mu.M) and noncompetitive vs. methylene blue. O2 inactivates the enzyme (t1/2 .apprx. 1 h) but is a reversible inhibitor of hydrogenase activity upon short exposure to low concentrations. Inhibition by O2 is uncompetitive vs. H2 and noncompetitive vs. methylene blue. Hydrogenase was not inhibited by C2H2; preincubation under C2H2 inactivates the enzyme. Reduced methyl and benzyl viologens support low rates of H2 evolution by the hydrogenase. The Km for reduced methyl viologen is 11 .mu.M. H2 is a potent inhibitor of H2 evolution: the inhibition is noncompetitive vs. reduced methyl viologen. The hydrogenase will catalyze a low rate of exchange in the reaction between D2 and H2O to form both HD and H2. A 2-site ping-pong mechanism for the enzyme in which H2 is reversibly activated at 1 site and e- carriers interact at the 2nd site.